Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

Abstract

Key WordsColumn liquid chromatography Glucoamylase Enantioselective retention and resolution Effect of operating parameters Ionic strength SummarySeveral mobile phase parameters were investigated for controlling enantioselective retention and resolution on a chiral stationary phase made in-house. The chiral selector was the enzyme amyloglucosidase, which was immobilized onto a silica support via reductive amination. The influences of the mobile phase pH, concentration and type of uncharged organic modifier, ionic strength and column temperature on enantioselectivity were studied. The analysis time for resolving enantiomers could be adjusted with only a minor decrease in enantiosetectivity by using a high ionic strength mobile phase buffer. This indicated a retention mechanism involving ion-exchange interactions. It was further confirmed by the decreasing enantioselectivity of amines when using a mobile phase pH below the isoelectric point of the native protein. Interesting effects were observed when the organic modifier concentration was increased and also when the column temperature was raised. Both retention and enantioselectivity increased with increasing concentration of 2-propanol in the mobile phase. Examples are given where both enantioselectivity and retention increased with increasing column temperature. Thermodynamic studies were performed to calculate the entropy and enthalpy constants. The results showed that, depending on mobile phase composition, the enantioselective retention may be caused by differences in entropy or enthalpy.

Chiral separation by chromatographic and electromigration techniques. A Review

“…Lysozyme [225]-and pepsin [226]-based CSPs showed enantioselectivity for basic and neutral enantiomers but not for acidic enantiomers. Recently, the preparation of amyloglucosidasebased CSPs and its application to aminoalcohols was reported [227]. Several protein-based CSPs are available from ChromTech AB, Sweden.…”

Separation of drug enantiomers by liquid chromatography and capillary electrophoresis, using immobilized proteins as chiral selectors

“…In native glucoamylase (G1 form) the catalytic domain (434 amino-acids) is connected to the starch-binding domain via an O-glycosylated region (residues 513-616). After binding to diol-silica, glucoamylase G1 displays chiral discrimination properties towards amino alcohols [30,146]. However, the enantioselectivity of metoprolol is lower on GA 1 columns compared to that obtained on CBH I CSPs [30,72].…”

Use of immobilized amyloglucosidase as chiral selector in chromatography. Immobilization and performance in liquid chromatography

“…Effects of solute structure on enantioselective recognition will also be discussed. However, the influence of several mobile phase parameters such as mobile phase pH, addition of organic modifiers and column temperature for controlling the enantioselective retention will be presented in another paper [ 16].…”